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Abstract

Background

Intestinal derived endotoxin and the subsequent endotoxemia can be considered major
predisposing factors for diseases such as atherosclerosis, sepsis, obesity and diabetes.
Dietary fat has been shown to increase postprandial endotoxemia. Therefore, the aim
of this study was to assess the effects of different dietary oils on intestinal endotoxin
transport and postprandial endotoxemia using swine as a model. We hypothesized that
oils rich in saturated fatty acids (SFA) would augment, while oils rich in n-3 polyunsaturated
fatty acids (PUFA) would attenuate intestinal endotoxin transport and circulating
concentrations.

Methods

Postprandial endotoxemia was measured in twenty four pigs following a porridge meal
made with either water (Control), fish oil (FO), vegetable oil (VO) or coconut oil
(CO). Blood was collected at 0, 1, 2, 3 and 5 hours postprandial and measured for
endotoxin. Furthermore, ex vivo ileum endotoxin transport was assessed using modified
Ussing chambers and intestines were treated with either no oil or 12.5% (v/v) VO,
FO, cod liver oil (CLO), CO or olive oil (OO). Ex vivo mucosal to serosal endotoxin
transport permeability (Papp) was then measured by the addition of fluorescent labeled-lipopolysaccharide.

Keywords:

Dietary fat; Endotoxin; Intestine

Background

The link between dietary fat and endogenous blood endotoxin has attracted increased
medical and biomedical interest over the last few years. Furthermore, hyperphagia,
increased adiposity and metabolic changes associated with high fat feeding can be
recapitulated in mice chronically infused with LPS for four weeks [1]. It has been reported that the structure of fat consumed (emulsion vs. free oil)
changes the extent of endotoxemia and that altering the composition, structure and
quality of dietary fats could improve health [2]. In healthy humans, postprandial plasma endotoxin concentrations increase on average
18% after a high fat meal (approximately 380 kcal from fat, 42% of total energy) compared
to the fasted state [3]. These authors concluded that increased postprandial LPS may contribute to the development
of postprandial inflammation and disease. Ghanim et al. [4,5] also showed that in healthy adults, high fat, high carbohydrate meal (~900 kcal)
increased postprandial plasma LPS concentrations by 70%. However, Laugerette et al.
[6] recently reported that dietary oil composition differentially modulated murine inflammation
and endotoxin transport. These authors also showed that fat composition, not quantity
in the diet (22 vs. 3%) was critical in modulating plasma endotoxemia. Collectively,
these data show that dietary fat intake and composition is able to modulate blood
endotoxin and that this is associated with acute inflammation and the metabolic diseases
of obesity and diabetes.

Both gram positive and gram negative bacteria are present in large quantities in the
intestine. Interestingly, the total quantity of endotoxin, which is the gram negative
bacterial outer cell wall component, in the intestine alone could be up to one gram
[7]. Even very small quantities of endotoxin, pico-gram scale, in the systemic circulation
has the potential to elicit an inflammatory response in humans and animals [8]. Endotoxin is also synonymously referred to as lipopolysaccharide (LPS), and both
of these compounds are major immunogens that elicit an inflammatory response in numerous
tissues and cell types via their recognition through pathogen-associated molecular
patterns (PAMPs) and Toll like receptors in the innate immune system [9]. Endotoxin is thought to enter circulation by transport across the intestinal epithelium
either via paracellular pathways through the openings of intestinal tight junctions
between two epithelial cells or by a transcellular pathway [7]. Transcellular transport and the associated endocytosis of intestinal derived endotoxin
may be facilitated by intracellular signaling processes mediated by the innate immune
receptor complex CD 14/Toll like receptor 4 (TLR4)/MD-2, in association with the cell
membrane micro domain lipid raft [10]. Furthermore, circulating endotoxin concentrations may also be augmented by transport
coupled to dietary lipids and chylomicrons [11].

In recent years accumulating research has investigated the link between dietary fat
and endogenous endotoxin in relation to metabolic inflammation [12,13]. Current evidence suggests that dietary fat augments circulating endotoxin concentrations
and the resultant postprandial endotoxemia leads to low-grade systemic inflammation
which has been implicated in the development of several metabolic diseases [1,3,14]. Intestinal derived endotoxin and the subsequent acute endotoxemia are considered
major predisposing factors for inflammation associated diseases such as atherosclerosis,
sepsis, obesity, type 2 diabetes and Alzheimer's [15-17]. However, the ability of different types of oil and fatty acids to facilitate uptake
of intestinal endotoxin has been poorly characterized. Interestingly, saturated and
n-3 polyunsaturated fatty acids (PUFA) have been shown to reciprocally modulate the
LPS receptor, TLR4, and cell membrane lipid rafts [18]. This is postulated to be due to saturated fatty acids (SFA) such as lauric and myristic
acid being part of the fatty acyl side chain composition of Lipid-A component of endotoxin
and the ability of n-3 PUFA to reduce the potency of endotoxin when substituted in
place of saturated fatty acids in lipid-A [19,20]. Thus, there is clear linkage between fatty acids (saturated, n-3 polyunsaturated,
monounsaturated etc. …) and endotoxin signaling.

Therefore, the aim of this study was to assess the effects of various dietary fats
on in vivo and ex vivo intestinal endotoxin transport and circulating concentrations
using the pig as a biomedical model. We hypothesize that oils rich in saturated fatty
acids (SFA) would augment, while the oils containing the n-3 PUFA (docosahexaenoic
acid [DHA] and eicosapentaenoic acid [EPA]) would attenuate, intestinal endotoxin
transport and postprandial endotoxemia.

Methods

Materials and animals

All the chemicals used for this study were purchased from Sigma-Aldrich (St. Louis,
MO) unless otherwise stated. All animal use and procedures were approved by the Iowa
State University Institutional Animal Care and Use Committee.

Effect of dietary oil on postprandial serum endotoxin concentration

Twenty four pigs (49 ± 7 kg BW) were raised on a typical corn-soybean diet that met
or exceeded their nutrient requirements [21] and randomly allocated to one of four treatments. The treatments consisted of 500 g
ground corn-soybean meal dough (2,145 kcal ME) made up with either 1) 50 ml water
(Control); 2) 50 ml fish oil (FO) (Spring Valley Inc., UT); 3) 50 ml vegetable oil
(VO) (Hy-Vee Inc., IA) ; or 4) 50 ml coconut oil (CO) (Spectrum Naturals Inc., NY).
After an overnight fast, six pigs were fed one of each porridge meal. Pigs voluntarily
consumed the entire porridge meal with in ten minutes after the meal was offered.
Blood was collected at 0, 1, 2, 3, and 5 hours postprandial by venipuncture using
pyrogen free vaccutainer tubes and sterile needles. Proper precautionary measures
were taken to prevent external endotoxin contamination of blood. Serum was separated
by centrifuging at 2000 × g and 4°C. Serum was then stored in pyrogen free tubes at
−80°C until further analysis.

Circulating serum endotoxin concentration was measured using the end point fluorescent
assay using the recombinant factor C (rFC) system (Lonza™, Switzerland). Briefly, the serum samples were diluted 1000 times and 100 μl of the
samples or standards were added to a 96 well plate and incubated at 37°C for 10 min.
Thereafter, 100 μL of rFC enzyme, rFC assay buffer and rFC substrate were added at
a ratio of 1:4:5 to the plate and an initial reading were taken followed by 1 h incubation
at 37°C. The relative fluorescence unit (RFU) for each well was determined (excitation
380 nm and emission 440 nm). A positive control standard from the assay kit was used
to ascertain the validity of the assay and the concentration of the endotoxin was
interpolated from the standard curve constructed from the standards and corrected
for sample dilution.

Ex vivo intestinal integrity and endotoxin transport

Freshly isolated ileum segments from eleven pigs (21–28 days old) were placed in chilled
Krebs-Henseleit buffer (consisting of, in mmol/L: 25 NaHCO3, 120 NaCl, 1 MgSO4, 6.3 KCl, 2 CaCl2, 0.32 NaH2PO4; pH 7.4) for transport to the laboratory while under constant aeration. Intestinal
tissues were then stripped of their outer serosal layer and immediately mounted into
modified Ussing chambers (Physiologic Instruments Inc., San Diego, CA and World Precision
Instruments Inc. New Haven, CT). Each chamber and intestinal segment (0.71 cm2) was bathed on its mucosal and serosal sides with 5 ml of Krebs-Henseleit buffer
and constantly gassed with 95% O2-5% CO2 mixture. Chambers were connected to a pair of dual channel current and voltage electrodes
containing 3% noble agar bridges and filled with 3 M potassium chloride to measure
electrophysiological parameters of the intestinal membranes or to measure the mucosal
to serosal transport of endotoxin. Transepithelial resistance (TER) was not different
across pigs, indicating no differences in paracellular permeability or leaky gut (data
not shown).

To rule out any influence that bile acids may have on intestinal integrity, TER and
macromolecule permeability was first tested on isolated ileum samples that were incubated
with porcine bile acid (0, 3, 6 and 9 mg/ml) for thirty minutes. Thereafter, FITC-labeled
dextran (FITC-Dextran, 4.4 kDa) mucosal to serosal transport was measured as described
previously [22]. Briefly, the mucosal chambers were challenged with 2.2 mg/mL FITC-Dextran and chamber
samples from both sides were collected every 10–15 min for eighty minutes. The relative
fluorescence was then determined using a fluorescent plate reader (Bio-Tek, USA) with
the excitation and emission wavelengths of 485 and 520 nm, respectively. Thereafter,
an apparent permeability coefficient (Papp) was calculated for each treatment:

The effect of dietary fat on endotoxin transport was studied using ex vivo permeability
of fluorescein isothiocyanate (FITC) labeled-LPS (Escherichia coli 055:B5) mounted
into modified Ussing chambers. Briefly, segments of swine intestinal tissues were
treated with either 12.5% (v/v) buffered saline control (CON), Fish Oil or cod liver
oil (CLO) manufactured by Spring Valley Inc., UT) or vegetable oil, coconut oil or
olive oil (OO) purchased form Hy-Vee Supermarkets Inc., IA). All oils were commercial
retail available and then mixed with 20 mM sodium taurodeoxycholate (bile acid) for
micelle formation to simulate the intestinal milieu. Each mucosal chamber was then
challenged with 20 μg/mL FITC-LPS and chamber samples were collected every 10–15 min
for eighty minutes. The relative fluorescence of each sample was then determined using
a fluorescent plate reader (Bio-Tek, USA) with the excitation and emission wavelengths
of 485 and 520 nm, respectively. The apparent permeability coefficient was then calculated
similar to that described above for FITC-Dextran.

Lipid rafts, dietary oil and ex vivo intestinal endotoxin transport

To examine the role of lipid rafts in intestinal endotoxin transport, ileum segments
from 16 pigs (56 ± 4 days of age) were mounted in Ussing chambers as described above.
Segments were pre-treated with or without 25 mM Methyl-β-cyclo dextrin (MβCD, a synthetic
lipid raft modifier) for 30 min. Thereafter, the mucosal chamber was spiked with either
saline-bile acid (CON) or Coconut oil-bile acid (12.5% v/v) and the FITC-LPS apparent
permeability coefficient for each tissue was calculated.

Fatty acid analysis

Fatty acid profiles of the dietary oils used to make the porridge were determined
and analyzed by GC-MS [23,24]. One ml oil was mixed with 0.5 mL of 4:1 hexane and 125 μg/L heptadecanoic acid was
added to each sample as an internal standard. FAME were analyzed by GC on a Hewlett-Packard
model 6890 fitted with an Omegawax 320 (30-m × 0.32-mm i.id. 0.25 um) capillary column.
Hydrogen was the carrier gas. The temperature program ranged from 80 to 250°C with
a temperature rise of 5°C/min. The injector and detector temperatures were 250°C and
1 μL of sample was injected and run split. Fatty acids methyl esters were identified
by their relative retention times on the column with respect to appropriate standards
and heptadecanoic acid.

Data analysis

Results are presented as means ± S.E.M and were analyzed with the Proc Mixed procedure
of SAS (Cary, NC). In the model, repetition or day of Ussing chamber run was used
as a random effect. Statistical significance of difference was analyzed by analysis
of variance (ANOVA) followed by Tukey’s range test for pair wise comparison of all
treatment means. Differences were considered significant at P ≤ 0.05 and a tendency
at P ≤ 0.10.

Effect of dietary oil on postprandial serum endotoxin concentration

To assess the effect of dietary lipids on postprandial serum endotoxin concentrations,
pigs received a porridge meal containing either 50 mL of saline, CO, VO or FO. The
endotoxin concentration of the various oils used did not differ (data not shown).
Change in postprandial serum endotoxin concentration due to different meal treatments
are presented in Figure 1A. The overall postprandial serum endotoxin concentrations were significantly lower
in the meals constituting saline or FO, with the mean overall serum endotoxin concentration
increasing two-fold over the saturated coconut oil meal treatment (P < 0.05, Figure 1B). However, meals made up with VO were not different from the saline, CO or FO treatments
(P < 0.05). Interestingly, the CO meal significantly elevated serum endotoxin concentrations
after 2 hours versus the saline and FO, and these remained elevated at 3 and 5 hour
postprandial (P < 0.05, Figure 1A).

Effect of exogenous porcine bile acid on ex vivo intestinal integrity

Bile acids have been shown to increase the intestinal permeability in cultured Caco-2
cell lines [25]. To rule out the effect that exogenous bile acid may reduce intestinal integrity,
freshly isolated pig ileum segments were used to measure TER (Figure 2A) and FITC-Dextran permeability (Figure 2B). As these segments were exposed to increasing concentrations of porcine bile ex
vivo, no differences in intestinal integrity were observed (P > 0.10, Figure 2). This might be due to the tolerance of intestinal tissues towards bile acid because
of previous exposure in vivo contrary to cell cultures where the cells are not exposed
to the bile acids previously.

Effect of dietary oil on ex vivo intestinal endotoxin transport

The ex vivo mucosal to serosal ileum endotoxin transport was assessed using modified
Ussing chambers and FITC-LPS permeability assay (Figure 3). Compared to the saline no oil control treatment, the endotoxin Papp was significantly
lower in both the FO and CLO treatments (P < 0.05). As hypothesized, the higher saturated
fat content of coconut oil significantly increased the endotoxin Papp compared to
the saline, FO and CLO (P < 0.05). However, mucosal treatment with VO and OO did not
differ from the saline or n-3 treatments (P > 0.05), but still attenuated endotoxin
Papp versus the coconut oil treatment (P < 0.05, Figure 3). Transepithelial resistance was not different due to ex vivo oil treatment (data
not shown).

To test the hypothesis that destabilization of intestinal lipid rafts would decrease
saturated fat induced endotoxin permeability, ileum samples were pretreated with the
lipid raft modifier methyl-β-cyclodextrin (MβCD) and coconut oil ex vivo. FITC-LPS
endotoxin transport permeability was then measured (Figure 4A). As expected, the CO treatment significantly augmented the ileum endotoxin Papp
compared to the saline control (P < 0.05). However, the endotoxin Papp was significantly
reduced with the MβCD treatment compared to the saline control (1.54 vs. 0.07, P = 0.04).
In the presence of MβCD and CO, the ileum Papp was attenuated three fold from the
CO alone treatment (P < 0.05). Importantly, ileum integrity and permeability as measured
by transepithelial resistance was not altered by either short term coconut oil, MβCD,
or the combination compared to the saline control (P = 0.98, Figure 4B).

Discussion

In Western diets, vegetable, canola and palm oils are common components of the diet
and to a lesser extent, long chain n-3 PUFA (DHA and EPA) oils from algal or marine
sources [26,27]. In recent years, the development of obesity, inflammation, atherosclerosis and other
metabolic diseases has been linked to low grade endotoxemia associated with high dietary
fat and energy intake [3,14,28-30]. However, these studies and others have raised questions on whether this diet induced
endotoxemia reflects changes in energy and fat content of the diet, intestinal permeability
or diet induced changes in gut microbiota. In the current study, we used ex vivo and
in vivo methods to examine intestinal permeability to endotoxin as it relates to dietary
oil composition. All pigs were clinically healthy and raised on typical commercial
swine corn-soybean diets. We observed no differences in intestinal integrity due to
our ex vivo treatments. Even though some cell culture experiments have been shown
to indicate bile acids affecting the intestinal permeability, we didn’t observe any
negative impacts from bile acids on intestinal integrity and permeability. Further,
Lang et al. reported that bile alone may not be sufficient to induce barrier function
in the Ex-vivo experiments [31]. Importantly, we only examined the acute actions of a meal or oil bolus treatment
and did not conduct a prolonged feeding trial in an attempt to change the pig microbiota
populations or the fatty acid profiles of tissues due to diet.

We hypothesized that dietary intake of oils rich in DHA and EPA would attenuate intestinal
endotoxin transport and postprandial circulating endotoxin. We found that dietary
cod liver and fish oils attenuated serum endotoxin concentrations compared to the
coconut oil and the endotoxin levels in these pigs were similar to the control group
(Figure 1). To the best of our knowledge, there are no other studies that have shown this effect
of DHA and EPA on endotoxin transport and blood endotoxemia.

Interestingly, only one paper has examined the effects of dietary oil composition
on endotoxin uptake and related inflammation [6]. However, contrary to our results, the report by Laugerette et al. [6] states that rape seed (canola) and sunflower oil, with its high unsaturated fatty
acid content, augmented plasma endotoxemia by 50-75%. Cani et al. [1], also observed a similar increase in endotoxemia in mice orally administered corn
oil with or without LPS compared to water alone. However, we observed no change in
serum postprandial endotoxin concentration or intestinal endotoxin transport due to
dietary vegetable oil compared to the saline control (Figures 1 and 3). This discrepancy of these parameters between the studies might be partially explained
by the use of different animal models and also the nature of the experimental design.
Whereas Laugerette et al. [6] performed a chronic feeding study using mice for eight weeks using oils with different
fatty acid composition; we used an acute meal bolus model with swine to examine endotoxin
permeability. Therefore, the mice fatty acid profiles would have mimicked their diets
and this tissue enrichment may also modify endotoxin permeability, signaling and acute
inflammation. Further work is needed to explain the molecular aspects of these differences
between the two studies with regard to acute versus chronic dietary fat signaling
in the gastrointestinal tract. Additionally, we observed a significant increase in
postprandial endotoxemia after a porridge meal mixed with coconut oil (Figure 1). Again, this contradicts data presented by Laugerette et al. [6] in which palm oil, high in saturated fatty acids, had no effect on plasma endotoxin
concentrations in mice. However, these authors did report an increase in plasma LPS
binding protein and argued that this protein is a better marker of endotoxemia due
to the short half-life of circulating endotoxin. One issue is that LPS binding protein
can be up regulated by inflammation and acute stress as well as both gram positive
and negative infections. As the magnitude of LBP response goes down with multiple
episodes of infection [32], this could be a result of the agonistic effects of saturated fatty acid on pro-inflammatory
signaling and not circulating endotoxin.

Gram negative bacteria, particularly those found in the distal ileum and colon, might
be one of the major sources for circulating endotoxin [33]. It has been estimated that a single cell of Escherichia coli contains approximately 106 Lipid A or endotoxin molecules and a typical human intestinal tract could harbor
approximately one gram of endotoxin [33-35]. Interestingly, the bacterial population in the intestine is not static. Multiple
studies have shown that bacterial composition shifts to either gram positive majority
or gram negative majority based on the composition of the diet consumed [30,36,37]. A majority of these studies show that consuming high saturated fat diet for a longer
period results in higher gram negative bacterial populations and high fiber diets
results in gram positive bacterial populations [30,38]. Laugerette et al. [6], reaffirmed this and showed that fatty acid composition of different dietary oils
can alter intestinal microbiota populations. Moreover, these authors demonstrated
that feeding a diet high in palm oil which is rich in SFAs significantly increased
the gram negative bacteria Escherichia coli groups, which can be significant source of endotoxin in the cecal content of mice
compared to milk fat, rape seed and sunflower oil fed diets.

During intestinal stress, ischemia, inflammation and diseases, paracellular transport
occurs through the tight junction, as known as “leaky gut” [39]. Alternatively, transcellular or intracellular transport can occur, particularly
in healthy individuals [40]. Transcellular endotoxin transported across a cell membrane has been shown to occur
via TLR4 and soluble GPI anchored receptor CD14 in a lipid raft mediated mechanism
[41,42]. Additionally, chylomicron associated LPS transport has also been suggested to play
a key role in intestinal LPS transport from the intestinal epithelial cell [11,43,44]. Importantly, we observed no decrease in intestinal integrity which might enhance
paracellular permeability as assessed by transepithelial resistance or FITC-dextran
permeability due to treatment or short term raft destabilization (Figure 4B). These data suggest that under healthy intestinal epithelial conditions, endotoxin
is most likely transported via lipid raft mediated endocytosis.

The signaling and transport process for endotoxin is initiated in specialized membrane
micro domains called lipid rafts [42]. Lipid rafts are membrane regions rich in cholesterol, glycolipids, sphingolipids
and saturated fatty acids, which result in a ‘rigid’ membrane structure compared to
the adjacent ‘fluid’ regions [45]. In immune cells, endotoxin triggers the recruitment of TLR4 into the lipid raft,
where it interacts with CD14 and other associated proteins such as MD-2 resulting
in an inflammatory signaling cascade [46,47]. Thus, the two major consequences of preventing endotoxin recognition by dissociating
the lipid raft to attenuate TLR4 recruitment include reduced inflammatory signaling
and attenuated endotoxin transport. We observed that if intestinal lipid rafts are
dissociated ex vivo with MβCD, then endotoxin permeability is attenuated in the ileum
(Figure 4). Interestingly, saturated fat induced endotoxin permeability is also significantly
reduced. Stimulation of TLR4 receptor has been shown to result in the endotoxin transport
across the intestinal epithelial cells [40]. TLR4 is not only implicated in the transcellular transport of LPS but also for live
bacteria [48]. Since saturated fatty acids and n-3 PUFA can reciprocally modulate TLR4 signaling
[49], the fatty acid composition of oil in the diet has the potential to increase or decrease
endotoxin transport. Altogether, these data suggest that apical endotoxin transport
in the intestines is arguably raft mediated in healthy individuals.

In vitro experiments show clearly that n-3 PUFA disrupt TLR4 signalling and the activation
of NFκB by LPS in a murine monocytic cell line [50]. Moreover, DHA modulates TLR4 signaling in vitro in RAW 264.7 macrophages and 293 T
cells [49], human monocytes and dendritic cells [51] and adipose tissue. We have previously shown in pigs that dietary EPA and DHA are
effective means of influencing the inflammatory status and pathways influenced by
TLR4 signaling induced by LPS [52] and in altering intestinal function [24,53]. Therefore, one could postulate that antagonizing TLR4 recruitment to lipid rafts
and it’s signaling by DHA and EPA, or stimulating these processes with saturated fatty
acids, would alter endotoxin transport and circulating postprandial endotoxin.

Another mechanism through which endotoxin can enter the circulation is through micelles.
Since the endotoxin side chains are made up of fatty acids, endotoxins can be incorporated
into the micelles and transported into the intestinal epithelial cell [54]. In intestinal epithelial cells, chylomicrons transport the absorbed lipids into
various parts of the body. High fat administration has been shown to proportionately
increase the endotoxin content of the chylomicron indicating that high fat consumption
indeed enhances higher endotoxin transport into the intestinal epithelial cell and
incorporation into chylomicron [11,28]. Furthermore, even though the mechanism is not clear, high intake of fat has been
shown to cause internalization of tight junction proteins and increase in the paracellular
permeability to macro molecules including endotoxin [30]. Even though, this mode of endotoxin transport cannot be ruled out, we speculate
that the rate of incorporation of fatty acids into micelles would not vary due to
oil composition. Therefore, we propose that the difference in intestinal endotoxin
transport we observed is primarily transcellular transport that involves lipid rafts
and receptor mediated endocytosis [42].

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VM, JH and NKG designed and conducted research presented. NKG was the principle investigator
and both NKG and JH obtained funding for this work. VM was the graduate student supervised
by NKG whom conducted most of the animal and laboratory work and wrote the manuscript.
JH and NKG supervised and revised the manuscript. All authors read and approved the
final manuscript.

Acknowledgements

This work was partially supported by funds acquired from the Iowa Pork Producers Association
and the Iowa State University Nutrition and Wellness Research Center. The authors
would like to thank Ms. Martha Jeffery and Yong Zhou for their assistance with the
animal and laboratory work presented in this project.